Impact of genomics and proteomics on Biotechnology / Biology

Genomics : Systemic study of the entire genome of an organism • To sequence the entire genome and to physically map the genome

arrangement (assign exact position of the genes /non-coding regions in genome)

• Before 1990s, the sequencing and study at a single gene level: Labo-rious and time-consuming task

• Development of high-throughput sequencing technologies and highly automated hardware system

Faster (in excess of 1 kb/h), cheaper, and more accurate Sequencing a human whole genome: ~ $ 1,000

Genome sequences of more than 2,000 organisms • Genomes of various animals and plants :, mouse, rat, sheep, pig,

monkey, dog, chicken, wheat, barley, Arabidopsis

Human genome project - Started in 1990 - Completed in 2003 : ~ 3.2 giga bases(Gb), 1,000 times larger than a typical bacterial genome - Less than 1/3 of the genome is transcribed into mRNA - Only 5 % of the RNA encodes polypeptides Number of polypeptide-encoding genes :~ 30,000

Provide full sequence information of every protein: - Identification of undiscovered proteins : Understanding their functions - Discovery of new drug targets Current drugs on the market target one of at most 500 proteins: Major targets are proteins (e.g., Kinases) Sequence data of many human pathogens (e.g., Helicobacter pylori, Mycobacterium tuberculosis, Vibrio cholerae) Provide drug targets against pathogens (e.g., gene products essential for pathogen viability or infectivity) Offer some clues in underlying mechanism of diseases Development of a new dug

New methods/tools in Biotechnology, Biology, and Medical sci-ences

Significance of genome data in Biotechnology/Biology

The ability to interrogate the human genome has altered our approach to studying complex diseases and development of therapies.

The emergence of genome-wide analysis tools has opened the door to investigating the function of each genes, genomic biomarker discovery, validation, and pharmacogenomics.

Leading medical/clinical researchers:• Actively studying genomic approaches to understand diseases,

and learn how these can be translated into medical and clinical settings.

Translational research

Issues

• Biological functions of between one-third and half of sequenced gene products remain unknown

• Assessment of biological functions of the sequenced genes Crucial to understanding the relationship between genotype

and phenotype as well as direct identification of drug targets

• Shift in the focus of genome research

Elucidation of biological function of genes

Functional genomics

• In the narrow sense : Biological function/activity of the isolated gene product

• In broader meaning : - Where in the cell the product acts, and what other cellular elements it interacts with

Interactome - How such interactions contributes to the overall physiology of the organism

Systems Biology

General definition of functional genomics :

• Determining the function of proteins deduced from genome sequence is a central goal in the post genome era • Elucidating the biological function of gene products

Assignment of function of gene products (Proteins)

• Biochemical (molecular) function• Assignment based on sequence homology• Based on structure• Based on ligand-binding specificity• Based on cellular process• Based on biological process• Based on proteomics or high-throughput

functional genomics

Conventional approaches

• Clone and express a gene to produce the protein encoded by the gene

• Try to purify the protein to homogeneity - Size, charge, hydrophobicity, oligomeric state, glycosylation

• Develop an assay for its function

• Identify the activity / function - Grow crystals, solve structure

Time-consuming and laborious for huge numbers of genes

Assignment of function to the sequenced gene products

Comparison of sequence/structure data in a high-through manner

Sequence homology study

• Computer-based sequence comparison between a gene of unknown function and genes whose functions (or gene product function) have been assigned• High homology : high similarity in function• Assigning a putative function to 40 - 60 % of all new gene se-

quences

Phylogenetic profiling

• A phylogenetic tree or evolutionary tree : a diagram showing the evo-lutionary interrelations of a group of organisms or genes or proteins derived from a common ancestral form based upon similarities and differences in their physical and/or genetic characteristics

• Youtube: www.youtube.com/watch?v=xwuhmMIIspo

• Phylogenetic profiling : Study of evolutionary relationships among var-ious biological species or other entities based on similarities and dif -ferences in their physical and/or genetic characteristics

• Closely related species should be expected to have very similar sets of genes

• Proteins that function in the same cellular context frequently have sim-ilar phylogenetic profiles : During evolution, all such functionally linked proteins tend to be either preserved or eliminated in a new species:

Proteins with similar profiles are likely to belong to a common group of functionally linked proteins.

• Establishing a pattern of presence or absence of a particular gene cod-ing for a protein of unknown function across a range of different organ-isms whose genomes have been sequences:

• Discovery of previously unknown enzymes in metabolic pathways, transcription factors that bind to conserved regulatory sites, and explanations for roles of certain mutations in human disease, plant specific gene functions

Rosetta Stone Approach

• Hypothesis: Some pairs of interacting proteins are encoded by two genes in some genome or by fused genes in other genomes

• Two separate polypeptides (X and Y) found in one organism may occur in a different organism as a single fused protein(XY)

• Function of the unknown gene in one organism can be deduced from the function of “fused genes” in different organism

Gyrase : Relieves strain while double-stranded DNA is being unwound by helicase

• Type II topoisomerase (heterodimer) : catalyzes the introduction of negative supercoils in DNA in the presence of ATP.

• Gyrase (bactrial topoisomerase II) : heterotetramer made up of 2 gyrA (97

kDa) subunits and 2 gyrB (90 kDa) subunits.

Knock-out animal study

• Generation and study of mice in which a specific gene has been deleted

• Phenotype observation

Structural genomics approach • Resolution of 3-D structure of proteins

Pathway maps Linked set of biochemical reactions catalyzed by enzymes• Questions:

– Is the extrapolation between species valid?– Have orthologs been identified accurately?

• Orthologs: Genes in different species that evolved from a common an-cestral gene by speciation, retaining the same function in the course of evo-lution.

- Identification of orthologs is critical for reliable prediction of gene function in newly sequenced genomes.

• Homologs : A gene related to a second gene by descent from a common ancestral DNA sequence regardless of their functions

• Paralogs: Genes related by duplication within a genome. Paralogs evolve new functions during a course of evolution, even if these are related to the original one.

DNA microarray technology : DNA chip Sequence data provide a map and possibility of assigning the putative

functions of the genes in genome based on sequence comparisons

Information regarding which genes are expressed and functionally active at any given circumstance and time

DNA microarray data : • Provide clues as to the biological function of the corresponding

genes Starting point

• Offer an approach to search for disease biomarkers and drug targets

ex) If a particular mRNA is only produced by a cancer cell compared to a normal cell, the mRNA (or its polypeptide product) may be a target for basic research for cancer, a good target for a new anti-cancer drug, biomarker for diagnosis.

DNA microarray (Gene chip) :• Comparison of mRNA expression levels between a sample (cancer

cell) and a reference (normal cell) in a high-throughput way : mRNA expression profiling - cDNA chip : mRNA expression profiling - Oligo chip ( ~ 50 mers) : mRNA expression profiling - SNPs (Single Nucleotide Polymorphisms) - Complementary probes are designed from gene sequence

Microarrays: Tool for gene expression profiling

• Solid support (such as glass microscope slide) on which DNA of known sequence (probes) is deposited in a grid-like array - 250,000 different short oligonucleotide probes in cm2

- 10,000 full-length cDNA in cm2

General procedure for mRNA expression profiling• mRNA is isolated from matched samples of interest.

• mRNA is typically converted to cDNA, labeled with fluorescence dyes(Cy3 and Cy5) or radioactivity

• Hybridization with the complementary probes on DNA chip • Analysis and comparison of expression levels of mRNAs between a sample and a reference mRNA expression profiling

Page 173

Microarrays: array surface

DNA Microarray Methodology - Flash Animation

www.bio.davidson.edu/Courses/genomics/chip/chip.html

Metazoans: human, mouse, rat, worm, insect

Fungi: yeast

Plants: Arabidopsis

Other organisms: e.g. bacteria, viruses

Organisms represented on microarrays

• Wild-type versus mutant cells

• Cultured cells with or without drug

• Physiological states (hibernation, cell polarity formation)

• Normal versus disease tissues (cancer, autism)

Questions to be addressed using microarrays

mRNA expression profiling using cDNA microarray

cDNA clones

PCR amplificationPurification

Robotic printing

sample reference

Reversetranscriptase

Hybridize targetsto microarray

Laser1 Laser2exitation

emission

Computeranalysis

mRNA

cy3 cy5

Green : up-regulated in a sample Red : up-regulated in a reference Yellow : equally expressed

Cy3 : ex 550 nm / em 570Cy5 : ex 649 nm / em 670

Commercially available DNA chip

purify RNA, label

hybridize,wash, image

Biological insight

Sampleacquisition

Dataacquisition

Data analysis

Data confirmation

data storage

experimentaldesign

Overall procedure

Stage 1: Experimental design

• Biological samples: technical and biological replicates

• mRNA extraction, conversion, labeling, hybridization

• Arrangement of array elements on a surface

PCR (Polymerase Chain Reaction)

Melting at 95 oCAnnealing at 55 oCElongation at 72 oC

Thermostable DNA polymerasefrom thermophilic bacterium

• Developed in 1983 by Kary Mullis• Nobel prize in Chemistry in 1993

Stage 2: RNA and probe preparation

• Confirm purity by running agarose gel

• Measure the absorbance at 260 and 280 nm and calculate the ratio to confirm purity and quantity

• Synthesis of cDNA and labeling using reverse transcriptase

Stage 3: Hybridization to DNA arrays

• Mixing of equal amounts of cDNA from a reference and a sample

• Load the solution to DNA microarray

• Incubation for hybridization followed by washing and drying

Stage 4: Image analysis

• Gene expression levels are quantified

• Fluorescence intensities are measured with a scanner, or radioactivity with a phosphorimage analyzer

Example of an approximately 37,500 probe-spotted oligo microarray with enlarged inset to show detail

Fig. 6.20Page 181

Stage 5: Microarray data analysis

• How can arrays be compared? • Which genes are regulated?• Are differences authentic?• What are the criteria for statistical significance?• Are there meaningful patterns in the data (such as groups)?

Stage 6: Biological confirmation

• Microarray experiments can be thought of as “hypothesis-generating” experiments : Clues

• Differential up- or down-regulation of specific genes can be confirmed using independent assays :

- Northern blots- Polymerase chain reaction (RT-PCR)- in situ hybridization

Use of DNA microarray

Comparison of gene expression levels

• Different tissues• Different environmental conditions (treated with drug)• Normal and cancer cells

• Search for a specific gene(s) responsible for biological

phenomenon Starting point for basic research

• Search for biopharmaceuticals/drug targets

• Identification of potential biomarkers for diagnosis

• SNP detection

Outcome of data analysis

But need validation

Rett

Control

Search for a gene responsible for a disease

A- B Crystallin is over-expressed in Rett Syndrome

Rett syndrome is a childhood neuro-developmental disorder characterized by normal early development followed by loss of purposeful use of the hands, distinctive hand movements, slowed brain and head growth, gait abnormalities, seizures, and mental retardation.

It affects females almost exclu-sively.

Gene Name Regulation

cytochrome b-561 DOWNTATA box binding protein (TBP)-associated factor, 32kDa DOWNglypican 4 DOWNAT rich interactive domain 4A (RBP1-like) DOWNTEA domain family member 4 UPG protein-coupled receptor 50 DOWNret finger protein 2 DOWNchromosome 11 open reading frame 24 UPnull DOWNnull DOWNnull UPnull DOWNminichromosome maintenance deficient 3 (S. cere-visiae) UPnull UPnull DOWNnull DOWNnull DOWNdefensin, alpha 6, Paneth cell-specific DOWNnull DOWNsmall nuclear ribonucleoprotein polypeptide C UPnull DOWNHLA-B associated transcript 3 UPmitogen-activated protein kinase kinase kinase 6 UPnull DOWN

Gene Name Regulation

alcohol dehydrogenase IB (class I), beta polypeptide DOWNmucolipin 1 DOWNnull UPcreatine kinase, brain DOWNartemin DOWNSP110 nuclear body protein DOWNapoptosis antagonizing transcription factor UPkiller cell lectin-like receptor subfamily D, member 1 DOWNfatty acid desaturase 3 DOWNSH2 domain protein 2A DOWNcholinergic receptor, nicotinic, epsilon polypeptide DOWNribosomal protein L29 UPTGFB-induced factor 2 (TALE family homeobox) DOWNectonucleoside triphosphate diphosphohydrolase 2 DOWNnull DOWNTBC1 domain family, member 8 (with GRAM domain) DOWN3-hydroxymethyl-3-methylglutaryl-Coenzyme A lyase (hydroxymethylglutaricaciduria) DOWNnull DOWNhomeo box D4 DOWNnull DOWNeukaryotic translation initiation factor 3, subunit 8, 110kDa UPRho guanine nucleotide exchange factor (GEF) 10 DOWNaquaporin 5 DOWN

Choi et al., J Thorac Oncol (2006), 1, 622-628

mRNA Expression Profiling in lung cancer patient and normal person using DNA Microarray

Expression profiles under different nutritional conditions

cDNA microarray chipcontaining 2,500 genes from yeast

• Expression profiling of genes from Yeast grown at 2% galac-tose and glucose

• Green: up-regulation in yeast grown at galactose• Red : up-regulation in yeast grown at glucose• Yellow : equally expressed

Lashkari et al., PNAS (1997)

Fast Data on >20,000 genes in several weeks

Comprehensive Entire yeast, mouse, and human genome on a chip

Flexible - As more genomes are sequenced, more arrays can be made.

- Custom arrays can be made to represent genes of interest

Easy Submit RNA samples to a core facility

Cheap Chip representing 20,000 genes for $350; Robotic spotter/scanner cost $50,000

Advantages of microarray experiments

Cost : Some researchers can’t afford to do appropriate controls, replicates

mRNA Final products of gene expression are proteinssignificance

Quality Impossible to assess elements on array surfacecontrol - Artifacts with image analysis

- Artifacts with data analysis

Disadvantages of microarray experiments

Proteomics Proteins are directly involved in most of biological functions• Drug targets : mostly proteins• Protein expression levels can not be accurately detected or mea-

sured via DNA array technology

mRNA levels are not directly correlated with those of the mRNA-encoded polypeptide

• A significant proportion of eukaryote mRNA undergoes differen-tial splicing, and can yield more than one polypeptide product

• No detailed information regarding how the functional activity of expressed proteins will be regulated.

(e.g., post-translational modifications : phosphorylation, glycosylation, sumoylation, proteolysis)

Proteomics : Proteom-based analysis

Proteins are responsible for specific functions, drug targets, or potential biomarkers :

More successfully identified by direct analysis of the expressed proteins in the cell

Systematic and comprehensive analysis of the proteins (pro-teom) expressed in the cell and their functions

- Direct comparison of protein expression levels - Changes in cellular protein profiles with cellular conditions

Proteomics approach by 2-D protein gels

General procedure

• Extraction of the total protein content from the target cell/tissue

• Separation of proteins by 2-D gel electrophoresis Dimension one: iso-electric focusing Dimension two: SDS-PAGE (polyacrylamide gel)

• Elution of protein spots • Analysis of eluted proteins for identification

2-D gel electrophoresis between two different conditions

How do you figure out which spot is what?

Protein micro-sequencing using Edman degradation protocol (partial amino acid sequence) : laborious and time-consuming

Protein analysis using mass spectrometry - Molecular mass of protein : MALDI-TOF - Digestion pattern by Trypsin : MALDI-TOF - Amino acid sequence of a digested peptide : Tandom mass spectrometry

Usually have a core facility do these, or collaborate with expert

Identification or assignment of protein function by sequence homol-ogy search

Basic components of a mass spectrometer

Convert sample molecules into ions (ionization)

Ion source Mass analyzer Detector

Sorts the ions by their masses by applying electromagnetic fields

Measures the value of an indicator quantity and thus provides data for calculating the abundances of each ion present

Mass spectrometers

Ion source Mass analyzer Detector

• MALDI (Matrix Assisted Laser Des-orption Ionization)

• ESI (Electrospray Ionization)• EI (Electron Ionization)• CI (Chemical Ionization)• FAB (Fast Atom Bombardment)

• TOF (Time of Flight)• Quadrupole• FT-ICR (FTMS)• Ion Trap

Time of Flight in mass spectrometry

• Ions are accelerated by an electric filed of known strength. - This acceleration results in an ion having the same kinetic energy as any other ion that has the same charge. - The velocity of the ion depends on the mass-to-charge ratio.

• The time for the ion to reach a detector at a known distance is measured.

- This time will depend on the mass-to-charge ratio of the ions. - The elapsed time from the instant a particle leaves a source to the instant it reaches a detector.

• From this time and the known experimental parameters, mass-to-charge of the ion is determined .

Time of Flight

• When the charged particle is accelerated into time-of-flight tube by the voltage U, its ki-netic energy of any mass is ½ mv2

• The smaller the molecular mass, the higher the velocity of a molecule ; Calculate the m/z by measuring the flight time

• Mass-to-charge (m/z) ratio of a molecule is determined by measuring the flight time in the tube

Matrix-assisted laser desorption/ionization (MALDI) • Soft ionization technique allows the analysis of biomolecules (such as pro-

tein, peptides, and sugars) and large organic molecules, which tend to be fragile and fragmented when ionized by more conventional ionization methods

• The matrix absorbs the laser energy, and the matrix is ionized (by addition of a proton) by this event.

• The matrix then transfers proton to the analyte molecules (e.g., protein molecules), thus charging the analyte

• Commonly used matrix - 3,5-dimethoxy-4-hydroxycinnamic acid(sinapinic acid), - α-cyano-4-hydroxycinnamic acid (alpha-cyano or alpha-matrix) - 2,5- dihydroxybenzoic acid (DHB)

MALDI-TOF Mass Spectrometry

Time-of-Flight mass spectrometry :

- Ions are accelerated by an electrical field to the same kinetic en-ergy

- The velocity of the ion depends on the mass-to-charge ratio. - From the elapsed time to reach a detector, the mass-to-charge

ratio can be determined.

Use of proteomics

Identification of a protein responsible for cellular function under specific conditions :

- Treatment of drugs, stress etc. - Identification of key enzymes in metabolic pathways - Construction of new strains

Discovery of disease biomarkers - Comparison of protein levels between patient and normal person Protein profiling

• Essential amino acid• Feed and food additives• Raw material for synthesis of various medicines

Amino acidProduction

(MT / annum)Capacity

(MT / annum)

L-Lysine-HCl 583,000 704,000

DL-Methion-ine 496,000 680,000

L-Threonine 27,000 49,000

World-wide production of amino acids

Source: Feedinfo. 2002

L-Threonine

Use of proteomics in metabolic pathway engineering

Biosynthetic pathway of L-Thr in E. coli

L-Aspartyl phosphate

Homoserine phosphate

Glucose

Phosphenolpyruvate

Pyruvate

TCA cycleOxaloacetate

ppc

mdh

aceBAKaspC

L-Lysine

L-Methionine

L-Aspartate

L-Aspartate semidaldehyde

Homoserine

L-Threonine

L-Isoleucine

thrA lysC

metL

asd

thrA

thrB

thrC

ilvA

dapA

metA

Feedback repression

Development of an L-Threonine-overproducing strain

Conventional mutagenic method

Use of protein expression profiles in biosynthetic path-way between parent and an L-threonine-producing strain

• Production level of L-threonine - W3110 (Wild-type E. Coli ) : < 0.001 g/L - TF 5015 (Mutant) : ~ 20 g/L

Proteome Analysis of two strains

W3110

TF5015

01234567

1.AldA

2.IcdA

3.AceA

4.ArgG

5. ThrC

6 7.OppA

8.LeuC

9. Udp

10.LeuD

11 12.YfiD

13 14

Protein

Expre

ssion

leve

l (arb

itrary

units

)

W3110TF5015

Lee et al., J Bacteriol (2004)

Identification of protein spots by MALDI-TDF